RNA Interference and Heterochromatic Silencing in Replication and Quiescence

复制和静止过程中的 RNA 干扰和异染色质沉默

• 批准号: 10330828
• 负责人: ROBERT A MARTIENSSEN
• 金额: $ 43.51万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-05 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10330828
• 关键词: Autoimmune Diseases; Biological Models; Bromodomain; Caenorhabditis elegans; Cell Cycle; Chromosomal Stability; Chromosome Segregation; Chromosomes; DNA; DNA Damage; DNA Polymerase II; DNA Repair; DNA replication fork; DNA-Directed RNA Polymerase; Disease; Drosophila genus; Epigenetic Process; Event; Fission Yeast; Gene Expression Regulation; Gene Silencing; Genetic Screening; Genetic Transcription; Genome Stability; Genomic Instability; Goals; Health; Heterochromatin; Hybrids; Link; Liquid substance; Malignant Neoplasms; Mammalian Cell; Mediating; MicroRNAs; Mus; Mutant Strains Mice; Pathway interactions; Phase; Phase Transition; Post-Transcriptional Regulation; Property; RNA; RNA Interference; Repetitive Sequence; Research; Ribonuclease H; Role; S phase; Structure; System; Transcription Coactivator; Ubiquitin; Untranslated RNA; Work; Yeasts; embryonic stem cell; epigenetic silencing; genome integrity; heterochromatin-specific nonhistone chromosomal protein HP-1; histone methylation; histone modification; homologous recombination; mutant; novel; piRNA; prevent; recruit; repaired; stem cell model; targeted treatment; ubiquitin ligase

Gene regulation by RNA interference is usually attributed to microRNA, but RNAi has a more ancient and fundamental role in heterochromatic silencing and genome stability. Heterochromatin comprises condensed repetitive regions of eukaryotic chromosomes, and mediates transcriptional silencing, chromosome segregation and genome integrity. We have found that heterochromatin is unexpectedly transcribed, and that subsequent RNAi guides histone modification. In the fission yeast S. pombe, “co- transcriptional” silencing occurs during the S phase of the cell cycle, followed by transcriptional silencing thereafter. Release of RNA polymerase II (Pol II) during replication prevents DNA damage, but without RNAi, replication forks stall and are repaired by homologous recombination (HR), causing genome instability. We have found that RNAi regulates genome stability through R-loops, RNA-DNA hybrid structures at transcription-replication collisions that promote HR. Silencing depends on histone modification, but recent studies show this may be an oversimplification. First, histone methylation recruits Heterochromatin Protein 1, which mediates liquid-liquid phase separation (LLPS) and may limit access to Pol II. Second, RNAi also recruits ubiquitin ligase, and we recently found that ubiquitin promotes these phase transitions. RNAi guides histone modification in C. elegans and Drosophila, but conservation in mammalian systems has been controversial. For example, piRNAs mediate histone modification in the germline but do not depend on Dicer, while genome instability in Dicer mutant mouse embryonic stem cells (mESC) depends on transcription of satellite repeats. Strikingly, we have found genome instability in Dicer-/- mESC depends on the transcriptional co- activator BRD4, and identical in-frame bromodomain deletions rescue Dicer mutants in fission yeast. S. pombe is an outstanding model system for cell cycle research, and we were the first to show that RNAi is essential for quiescence (G0). Genetic screens have revealed that nucleolar RNA silencing and histone modifications mediate this novel function. Our goals in the next five years are to determine the elusive mechanism by which RNAi guides each aspect of heterochromatin from repeat instability, to silencing, chromosome segregation and DNA repair as well as survival in quiescence. Our recent work suggests a central role for long non-coding RNA, R-loops and the activity of RNAse H in the upstream steps in this pathway. Downstream events include histone modification and LLPS that may underlie the classically “condensed” properties of heterochromatin. We will use our stem cell model to assess the conservation and relevance of these mechanisms in health and disease, especially in cancer.

RNA干扰的基因调控通常归因于microRNA，但RNAi有更古老的 以及异染色质沉默和基因组稳定性中的基本作用。异染色质包括 真核染色体的浓缩重复区域，并介导转录沉默， 染色体分离和基因组完整性。我们意外地发现异染色质 转录，随后的 RNAi 指导组蛋白修饰。在裂殖酵母中，“co- “转录”沉默发生在细胞周期的 S 期，随后是转录沉默 此后。复制过程中释放 RNA 聚合酶 II (Pol II) 可防止 DNA 损伤，但不 RNAi、复制叉停滞并通过同源重组 (HR) 修复，导致基因组 不稳定。我们发现RNAi通过R环、RNA-DNA杂合体调节基因组稳定性 促进 HR 的转录复制碰撞结构。 沉默取决于组蛋白修饰，但最近的研究表明这可能过于简单化。 首先，组蛋白甲基化招募异染色质蛋白 1，它介​​导液-液相 分离 (LLPS)，并可能限制对 Pol II 的访问。其次，RNAi 还招募泛素连接酶，我们 最近发现泛素促进这些相变。 RNAi 指导 C 中的组蛋白修饰。 线虫和果蝇，但哺乳动物系统中的保护一直存在争议。例如， piRNA 介导种系中的组蛋白修饰，但不依赖于 Dicer，而基因组 Dicer 突变型小鼠胚胎干细胞 (mESC) 的不稳定性取决于卫星的转录 重复。引人注目的是，我们发现 Dicer-/- mESC 中的基因组不稳定性取决于转录共 激活剂 BRD4 和相同的框内溴结构域缺失拯救了裂殖酵母中的 Dicer 突变体。 粟酒裂殖酵母是细胞周期研究的杰出模型系统，我们是第一个证明这一点的人 RNAi 对于静止 (G0) 至关重要。遗传筛选表明核仁 RNA 沉默和 组蛋白修饰介导了这一新功能。我们未来五年的目标是确定 RNAi 引导异染色质各个方面从重复不稳定到 沉默、染色体分离和DNA修复以及静止状态下的存活。我们最近的工作 表明长链非编码 RNA、R 环和 RNAse H 活性在上游发挥着核心作用 在此路径中的步骤。下游事件包括组蛋白修饰和 LLPS，它们可能是 异染色质的经典“浓缩”特性。我们将使用我们的干细胞模型来评估 这些机制在健康和疾病中的保护和相关性，特别是在癌症中。